A. Taxol and the production of secondary plant metabolites in vitro
Taxol, a diterpene, was first identified in 1964 and has subsequently been shown to have anti-cancer activity against ovarian cancer, breast cancer, small-cell lung cancer, melanoma, and colon cancer.
Taxol is produced primarily in the bark and cambial tissue of the pacific yew Taxus brevifolia. Using current purification procedures, 1 kilogram of taxol requires processing of approximately 10,000 kilograms of bark. This is equivalent to 2,000-4,000 sixty to seventy year old trees. Recent estimates put the need of taxol at approximately 250 kilograms of the purified drug per year. This is equivalent to a yield of 25 million kilograms of dried bark or approximately 750,000 trees. Due to the shortage of the pacific yew, other sources of taxol are currently being sought.
One potential source of taxol which has been examined is in vitro cultured plant cells and tissues. U.S. Pat. No. 5,019,504 describes the initiation and proliferation of callus cell cultures from explants of T. brevifolia. The callus cells produced by this procedure were shown to produce taxol.
There are several obstacles to the use of callus or undifferentiated cell cultures as a means of producing secondary metabolites such as taxol. Typically, secondary metabolites are produced by specialized or differentiated tissues; most notably bark in the case of taxol, or leaves in the case of other taxanes such as baccatin. Undifferentiated, or callus cultures often lack the necessary biosynthetic capacity to assemble molecules as complex as taxanes, or, the degree of cytodifferentiation required to sequester these molecules once synthesized. The result has been that most secondary metabolites are not found in callus cultures, and, in cases where they have been detected in callus, the concentration is usually very much lower than that in planta.
Nevertheless, callus cultures have been extensively investigated for the production of secondary metabolites due to their ease of establishment, manipulation and rapid growth rate. Often, for investigations in vitro, rapidly-growing callus cultures are the most convenient way to produce the large quantities of tissue required for detection of secondary metabolites such as taxol that are found in such low concentrations. The rapid growth rate of callus cultures underscores another of their disadvantages in that the cells that comprise these cultures tend to be genetically unstable, demonstrating high levels of genetic recombination and unstable ploidy levels. Such genetic instability can ultimately lead to cultures with diminished taxol production capacity. In order to avoid this problem, callus cultures need to continually be reestablished from a genetic stock.
For the large scale production of secondary plant products in vitro, it would be desirable to combine the rapid growth rates and capacity for high biomass concentrations of undifferentiated cell culture systems with the inherent capacity for secondary metabolite production of differentiated cells or tissues. Researches have realized the potential for secondary metabolite production in cultures that proliferate in a manner akin to undifferentiated cell cultures (i.e., callus or cell suspension), but are instead comprised of differentiated cells or tissues. For example shoot cultures (tissue cultures comprised of masses of rapidly proliferating shoots) have been investigated as sources of essential oils and alkaloids that are found in leaf or stem tissue (Heble, in Primary and Secondary Metabolism in Plant Cell Cultures, Neumann et al. (ed.), Springer-Verlag, Berlin Heedelberg, pp.281-289 (1985)). In shoot cultures, the specific tissue types that produce and sequester essential leaf oils are multiplied, and the rigid developmental program required for shoot morphogenesis also minimizes genetic instability. In this way, tissue cultures have been shown to combine the attractive features of both undifferentiated and differentiated systems.
Although taxol has been detected in undifferentiated cell culture systems, its production has not been described in tissue culture systems. One such system is embryogenic tissue cultures.
Embryogenic conifer tissue cultures are strikingly dissimilar to conifer callus cultures biochemically, histologically, and in macroscopic appearance. Although the term "callus" is a generic term used to describe cell and tissue cultures, many researchers in the field of conifer somatic embryogenesis object to the use of "callus" in describing embryogenic conifer tissue (See for example Gupta and Durzan, Bio/Tech. 5:147-151 (1987); Rohr et al., Amer. J. Bot. 76:1460-1467 (1988); Tautorus et al., Can. J. Bot. 69:1873-1899 (1991)). The reason for the objection to the use of the term "callus" is that, rather than being comprised of undifferentiated cells, embryogenic conifer tissue cultures are comprised of differentiated cells (suspensor-like cells) and structures analogous to early stage embryos found in developing seeds. Therefore, embryogenic conifer tissue cultures do not fit the detention of callus or their liquid counterparts, cell suspension cultures, and represent an improved way to produce taxol by the embodiment of the beneficial growth characteristics of cell culture systems with the capacity for secondary metabolite production of tissue culture systems.
B. Somatic Embryogenesis in Conifers
Although procedures for the induction of somatic embryogenesis have been known in the art for some time (Tisseral et al,. Hort. Re.1:1-78 (1979)), it has only been recently demonstrated successfully with coniferous species (See Hakman et al. Plant Sci. 38:53-59 (1985)). Since the first reports of successful induction of somatic embryogenesis in conifer cell cultures, eighteen (18) species from the genera Pinus, Picea, Abies, Larix, and Psuedotsuga (Tautorus et al., Can. J. Bot. 69:1873-1899 (1991)), have been demonstrated as having the capacity to produce somatic embryos.
The production of somatic embryos from conifers is not universal. Several important varieties have yet to be successfully cultured, such as members of the genus Taxus.
C. Transgenic Plants.
Recent advances in recombinant DNA and genetic technologies have made it possible to introduce and express a desired gene sequence in a recipient plant. Through the use of such methods, plants have been engineered to express gene sequences that are not normally or naturally present in the native plant, or to exhibit altered expression of naturally occurring genes. Plants produced through the use of recombinant techniques are known as "transgenic" plants.
Transgenic plants are generally produced by transforming a single plant cell and then regenerating a whole plant from the cell via somatic embryogenesis. Since many genera of plants have been regenerated from a single cell (Friedt, W. et al. Prog. Botany 49:192-215 (1987); Brunold, C. et al., Molec. Gen. Genet. 208:469-473 (1987); Durand, J. et al., Plant Sci62:263-272 (1989); Attree et al., Can. J. Bot. 67:1790-1795 (1989)), successful production of transgenic plants from a wide variety of plant groups is theoretically possible.
Several methods have been developed to deliver and express a foreign gene into a plant cell. These include engineered Ti plasmids from the soil bacterium A. tumefaciens (Czako, M. et al., Plant Mol. Biol. 6:101-109 (1986); Feirer et al., Proceedings 20th Southern Forest Tree Improvement Conference, Jun. 26-30, 1989, Charleston, S.C., pg. 381; Jones, J. D. G. et al., EMBO J. 4:2411-2418 (1985), engineered plant viruses such as the cauliflower mosaic virus (Shah, D. M. et al., Science 233:478-481 (1986)); Schewmaker, C. K. et al., Virol. 140:281-288 (1985)), microinjection of gene sequences into a plant cell (Crossway, A. et al., Molec. Gen. Genet. 202:179-185 (1986); Potrykus, I. et al., Molec. Gen. Genet. 199:169-177 (1985)), electroporation (Fromm, M.E. et al., Nature 319:791-793 (1986); Tautorus et al., Theor. Appl. Genet. 78:531-536 (1989), and DNA coated particle acceleration (Bolik, M. et al. Protoplasma 162:61-68 (1991)). Several of these procedures have been successfully employed to transform conifer tissues in vitro. (Ellis et al., International Society of Plant Molecular Biology, meeting of Oct. 6-11, 1991, Tucson, Ariz.).